Animal Cell Culture
Differences between procaryotes and eucaryotes
Eucaryotes
size
10-30 um
spherical,
shape
ellipsoidal
locomotion
no
border
membrane
Procaryotes
1-2 um
rods, ellipses,
etc.
yes
wall
Cells are negatively charged.
• attach to positively charges surfaces
• some cells must attach to grow, others not
• examples of surfaces: sephadex, collagen
• positively charged vesicles containing stuff you would like to put into
the cell will attach to cell surfaces and be taken into the cell
Cell Components of note…
Endoplasmic reticulum
• network of membrane-bound channels
• channels: outside is the cytosol, inside are the ribosomes
• e.r. responsible for the protein synthesis and post-translational processing
Mitochondria:
• respiration occurs there -> ATP produced
• independent organelles with their own DNA, capable of independent
reproduction
• two membranes
• smooth outer membrane
• folded inner membrane called the cristae
Lysosomes:
• small organelles, single membrane
• contain hydrolytic enzymes: proteases, nucleases and esterases
Golgi body:
• collection and secretion of extracellular proteins
• direct intracellular protein traffic
Peroxisomes: contain peroxidases – hydrolyze H2O2
Glyoxysomes: glyoxalases (glyoxylic acid metabolism)
Nucleus:
• two nuclear membranes – form a nuclear envelope
• nuclear pores – continuity between nucleus and cytoplasm
• chromosomes and histones
Growth Medium
• glucose, glutamine, amino acids,
• serum: liquid extracted from blood of offspring removed from freshly-killed
pregnant cows.
• proteins: cell attachment factors; metal binding proteins; protease inhibitors
• peptides: various growth factors
• hormones: stimulate growth and nutrient uptake
• nutrients
• metabolites
• minerals
Metabolism
Glucose -> pyruvate via glycolysis
Glucose -> biosynthesis via pentose phosphate pathway
Pyruvate -> CO2, H2O, via TCA cycle
Pyruvate -> lactic acid, fatty acids
Glutamine is a carbon and energy source:
1.
Deaminated to glutamate-> other amino acids
2.
Enters TCA cycle for other amino acids, etc.
• Animal cells can
synthesize glucose from
pyruvate via
gluconeogenesis pathway
• waste: lactate,
ammonia
• at high levels, these
are toxic
• challenge for high
density cultures
Cultivation of Animal Cells
1.
Tissues are removed from animals and transferred to growth medium
2.
Organs -> lung, kidney, etc. (cells grow attached)
3.
These are primary cultures
4.
Cells can be transferred to new flasks once they have grown into a
monolayer
•
Remove cells with a protease – trypsin, collagenase, pronase or EDTA
•
Wash cells with serum containing medium (centrifuge gently)
•
Resuspend in growth medium
•
Plate onto a fresh flask
Differentiated mammalian cells are mortal, however, cancer cell lines are
immortal.
Animal cell lines include: mammal, insect, fish, crustaceans
Insect cells are easier to grow. They grow faster and you can use a
baculovirus as a vector for genetic engineering. Insect cells may not have
post-translational modifications like mammalian cells.
The blastocyst has an outer layer of cells and inside the hollow
sphere, there is a cluster of cells called the inner cell mass.
Outer cell layer  placenta and supporting tissues for fetus in uterus
Inner cell mass
 all other body tissues
 pluripotent
Umbilical Cord Stem Cells
Abundant source of the stem cells that produce blood and
immune cells.
Adult Stem Cells
Found in the brain, bone marrow, blood, skeletal muscle, skin,
fat and elsewhere. They are difficult to identify, isolate and grow
in culture, and they may not be as versatile as embryonic stem
cells. But then again, what is research all about?
Commonly used cell lines
• Chinese Hamster Ovary cells: CHO cells
• HeLa cells
• mouse kidney cells
Commonly used medium
• nutrients + 5-20% serum ($100-$500 per liter)
Problems with serum
• cost
• virus – safety issues
• extra-cellular proteins
• lot-to-lot variation
• availability
• foaming
Book: serum-free media contains insulin, transferrin, fibronectin, other protein
components
Serum-free media can also be protein-free
Hybridoma Cells
• antibody-producing lymphocytes fused with cancer cells – myeloma
• lymphocytes grow slowly and are mortal, hybridoma cells are immortal and
produce antibodies
Production of antibody
fragments by fungi and bacteria
See Nyyssonen and Eini;
Bio/Technology 1993 vol 11(5) p.
591.
Kinetics of growth
Similar to bacterial culture
There is a difference between attached and suspended cultures
Disposable bioreactors
Cell growth measured by actual cell counts
“Hemocytometer”
Stain cells and drop on the slide – count all the white ones
Cell growth is measured in days.
Production can continue in non-growth conditions – hopefully!
Oxygen requirements: .06 - .2 x 10-12 mol O2/h/cell OUR ~ 0.1-0.6 mmol O2/l/hr
Compare to bacteria at 10 – 200 mmol/l/hr!
Animal Cells are shear sensitive – cannot sparge reactors
• cells respond to shear with apoptosis
Fritted metal fittings create very small bubbles
Chemical (e.g. Pluronic F-68) can be added to provide shear protection
Typical kLa of suspension cultures (106 cells/ml) 5 – 25 hr-1
Bioreactor Considerations for Animal Cell Culture
Microcarriers: sephadex, etc: 70,000 cm2/liter: get ~ 107 cells/ml
• cells grow in mono – multi-layers on microcarriers
Hollow Fiber reactors
• cells grow on the outside of the tubes, nutrients pass through the tubes
• uncontrolled, unmixed environment
• get high cell concentrations (eg hybridoma demonstrated at 5 – 50
mg/ml antibody
Stirred-Tank reactors
• use pitched blade or other impeller (10 – 30 RPM for stirrers)
•Tank and bubble columns are used (especially with cells on multicarriers)
Perfusion reactor
• simultaneous cell cultivation and product concentration and byproduct removal
• sonic separator
Products from Animal Cell Cultures
1. Immunobiologicals:
monoclonal antibodies
immunobiological regulators
a)
Used for diagnostic assay systems, therapeutics for biological separation
systems (affinity chromatography)
b)
Interferon
2.
Virus Vaccines
3.
Hormones: glycosylated peptides (e.g. erythropoetin)
4.
Enzymes: TPA, collagenase,factor VII, factor VIII, factor X